Quantitative Analysis of Iris Parameters in Keratoconus Patients

Original Article

Quantitative analysis of iris parameters in keratoconus patients using optical coherence tomography Uso de tomografia de coerência óptica na análise quantitativa dos parâmetros irianos em pacientes com ceratocone

Gustavo Bonfadini1,2,3, Karun Arora4, Lucas M. Vianna1,3,5, Mauro Campos3, David Friedman4,6, Beatriz Muñoz4,6, Albert S Jun1

ABSTRACT RESUMO Purpose: To investigate the relationship between quantitative iris parameters Objetivo: Investigar a relação entre os parâmetros quantitativos irianos e a presença and the presence of keratoconus. de ceratocone. Methods: Cross-sectional observational study that included 15 affected eyes of Métodos: Estudo observacional transversal com quinze olhos de 15 pacientes com 15 patients with keratoconus and 26 eyes of 26 normal age- and sex-matched ceratocone e 26 olhos de 26 indivíduos normais, pareados por idade e gênero. Parâ- controls. Iris parameters (area, thickness, and pupil diameter) of affected and metros da íris (área, espessura e diâmetro da pupila) de olhos com ceratocone e olhos unaffected eyes were measured under standardized light and dark conditions sem ceratocone foram medidos usando tomografia de coerência óptica do segmento using anterior segment optical coherence tomography (AS-OCT). To identify opti­ anterior (AS-OCT), em condições padronizadas de alta luminosidade e ambiente mal iris thickness cutoff points to maximize the sensitivity and specificity when escuro. Com o objetivo de maximizar a sensibilidade, especificidade e identificar o discriminating keratoconus eyes from normal eyes, the analysis included the use melhor ponto de corte na diferenciação entre ceratocone e indivíduos normais, foi of receiver operating characteristic (ROC) curves. realizada a análise quantitativa da curva característica operacional do receptor (ROC) Results: Iris thickness and area were lower in keratoconus eyes than in normal dos parâmetros de espessura da íris. eyes. The mean thickness at the pupillary margin under both light and dark condi- Resultados: A área e espessura da íris estavam reduzidas nos olhos de pacientes com tions was found to be the best parameter for discriminating normal patients from ceratocone. Observamos que o melhor parâmetro para discriminar indivíduos normais keratoconus patients. Diagnostic performance was assessed by the area under the de pacientes com ceratocone foi a espessura média na margem pupilar, tanto em ROC curve (AROC), which had a value of 0.8256 with 80.0% sensitivity and 84.6% condições de alta luminosidade quanto em ambiente escuro. O desempenho diagnós- specificity, using a cutoff of 0.4125 mm. The sensitivity increased to 86.7% when tico deste parâmetro foi avaliado pela análise quantitativa da área sob a curva ROC a cutoff of 0.4700 mm was used. (AROC), mostrando AROC de 0,8256, com sensibilidade de 80,0% e especificidade de Conclusions: In our sample, iris thickness was lower in keratoconus eyes than 84,6% usando um ponto de corte de 0,4125 milímetros de espessura da íris. A sensibi- in normal eyes. These results suggest that tomographic parameters may provide lidade aumentou para 86,7%, com um corte de 0,4700 milímetros de espessura da íris. novel adjunct approaches for keratoconus screening. Conclusões: Na amostra deste estudo, a espessura da íris demostrou-se reduzida nos olhos com ceratocone. Estes resultados sugerem que os parâmetros tomográficos podem proporcionar novas informações no auxilio da triagem de pacientes com ceratocone. Keywords: Tomography, optical coherence; Iris; Keratoconus; Cornea; Dilatation Descritores: Tomografia de coerência óptica; Íris; Ceratocone; Córnea; Dilatação pa­­ pathologic; ROC curve tológica; Curva ROC

INTRODUCTION plantation has inherent intra- and postoperative risks, and the surgery Keratoconus is traditionally described as a noninflammatory ectatic significantly affects the patient’s quality of life during the surgical re- disorder of the cornea characterized by progressive thinning, steepe- covery phase and beyond, with lost work time and often permanent ning, and apical protrusion. These corneal changes induce irregular changes in vision and lifestyle. astigmatism and myopic shift, causing impairment of vision. The The diagnosis of moderate-to-advanced keratoconus is not diffi­ incidence of keratoconus in the general population is estimated to be cult because of the presence of irregular astigmatism on corneal approximately 1 in 2000(1). Keratoconus is the second most frequent topography and the development of classical clinical signs. However, indication for corneal transplantation, accounting for approximately identifying subclinical forms of the disease in patients with normal 12% of corneal transplants performed in the United States(2) and is best-corrected visual acuity (BCVA) and minimum or no clinical signs one of the main indications of keratoplasty in Brazil(3). Corneal trans- is challenging. The identification of very early forms of keratoconus

Submitted for publication: February 23, 2015 Funding: No specific financial support was available for this study. Accepted for publication: July 6, 2015 Disclosure of potential conflicts of interest: None of the authors have any potential conflict of 1 Cornea and Anterior Segment Service, The Wilmer Eye Institute, Johns Hopkins School of Medicine, interest to disclosure. Baltimore, MD, USA. 2 Corresponding author: Albert S. Jun. 400 N. Broadway, Smith Building 5011 - Baltimore, MD Rio de Janeiro Eye Bank (INTO), Rio de Janeiro, RJ, Brazil. 21231 - E-mail: [email protected] 3 Department of Ophthalmology and Visual Sciences, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil. Approved by the following research ethics committee: Institutional Review Boards (IRB): 4 Dana Center for Preventive Ophthalmology, The Wilmer Eye Institute, Johns Hopkins School of No 00051452. Johns Hopkins School of Medicine, Baltimore, MD, USA. Medicine, Baltimore, MD, USA. 5 Universidade Estadual do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 6 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.

http://dx.doi.org/10.5935/0004-2749.20150080 Arq Bras Oftalmol. 2015;78(5):305-9 305 Quantitative analysis of iris parameters in keratoconus patients using optical coherence tomography

or forme fruste keratoconus relies on topographic data and subjec- Table 1. Participant characteristics tive clinical impression. Thus, more objective parameters are needed Keratoconus Controls when evaluating patients with forme fruste keratoconus, such as n=15 n=26 those with asymmetric or unilateral keratoconus, family members Parameter Mean (SD) Mean (SD) P-value of keratoconus patients, and patients undergoing evaluation for re­ fractive surgery. Demographics Corneal tomography provides three-dimensional reconstruction Age in years 38.0 (11.4) 34.1 (10.0) 0.260 of the cornea, enabling evaluation of the anterior and posterior corneal Male 77.7% 46.2% 0.200 surfaces and creation of a corneal pachymetric (thickness) and topo- Race graphic map. Commercially available corneal tomography systems White 46.7% 61.5% *Fisher’s exact that use a rotating Scheimpflug cameras have been proposed to 0.004 help identify forme fruste keratoconus(4), and high-speed anterior Asian 00.0% 19.2% segment optical coherence tomography has been shown to have Black 46.7% 03.9% good repeatability and reproducibility with ocular structures and iris Hispanic 06.7% 07.7% area measurements(5-9). Refraction In a subset of keratoconus patients undergoing penetrating kera- toplasty (PKP), we have observed that the iris can show a tendency to Sphere -3.5 (2.1) -5.2 (3.3) 0.080 prolapse toward the incisions and be less responsive to pharmacolo- Cylinder -3.0 (2.4) -0.7 (0.8) 0.030 gical agents. Urrets-Zavalia syndrome, an uncommon postoperative Spherical equivalent -2.0 (2.4) -4.8 (3.0) 0.004 complication of PKP, has also been associated with keratoconus, which SD= standard deviation. suggests an intrinsic abnormality in irises with this disease(10). Others *= Fisher’s exact test was used to compare the racial distribution between keratoconus have described an association between keratoconus and floppy patients and controls. eyelids(11,12). Thus, multiple lines of evidence indicate extracorneal ma­­ nifestations of keratoconus involving the iris and/or the periocular skin and connective tissues. In this report, we describe the novel use of AS-OCT to determine iris parameters for discriminating between normal individuals and AS-OCT image acquisition keratoconus patients. The Visante OCT used in this study has axial and transverse image resolution up to 18 and 60 μm, respectively. All participants were METHODS imaged in the enhanced anterior segment single mode to acquire an image centered over the pupil on the horizontal meridian, and Subjects proper eye alignment was indicated by an interference beam along Approval was granted by the Johns Hopkins Institutional Review the visual axis (scan length 16 mm; 256 A-scans). No pharmacologic Board for this study. All study procedures adhered to the tenets of agents were applied to the eye prior to imaging. The inset image the Declaration of Helsinki. This was a prospective, cross-sectional, on the monitor screen was used as a guide to ensure that there was clinic-based evaluation of patients who presented to the cornea minimal eye movement and no blinking during imaging. service at the Wilmer Eye Institute. The study population consisted AS-OCT imaging of the study eye was first obtained in a com- of recruited patients with a known or new diagnosis of keratoconus pletely darkened room with all room lights switched off (<1 lux and healthy controls with normal myopic refraction. illumination). We allowed 3 min for patients’ dark adaptation before Inclusion criteria were as follows: (1) willingness and ability to par- image acquisition. Repeat AS-OCT imaging under illumination was ticipate; (2) persons aged between 18 and 60 years; and (3) no ocular acquired 1 min after the room lights were turned on (350-400 lux). A surgery in the studied eye. Exclusion criteria were as follows: (1) pre- fully charged handheld torch kept at an approximate angulation of sence of any anatomic iris abnormalities, such as peripheral anterior 45o and a distance of 25 cm was then shone from the temporal aspect synechiae or iris whorling (distortion of radially orientated iris fibers); onto the fellow eye (light intensity at the subject’s sitting position was (2) any prior intraocular incisional surgery or laser treatment; (3) any 1600 lux) during the entire time of the AS-OCT image acquisition. factor limiting normal AS-OCT imaging of the anterior segment; (4) Care was taken to prevent any light from crossing the nasal bridge. history of topical or systemic medications that could interfere with iris At least three images under dark and light conditions were acqui- anatomy or physiology; (5) history of any neurological disease; and red in each study eye. The image with the best quality, defined as ha- (6) presence of glaucoma, elevated intraocular pressure (IOP) above ving good visible scleral spurs (SS) at the temporal and nasal angles, 20 mmHg, or “glaucomflecken” lens opacity. no movement artifacts (i.e., no discontinuity of the anterior segment All participants completed a comprehensive questionnaire desig- structures in the AS-OCT image), and good centration, was selected ned to collect information on baseline demographics, ocular history, for analysis from the dark and light series of images, respectively. eye rubbing, contact lens use, past medical history, and family history.­ Participants underwent a complete ophthalmic examination, inclu- Image analysis ding measurement of BCVA, spherical equivalent refraction in diop- Iris parameters on each of the two images (dark and light condi- ters (D), slit lamp biomicroscopy, and AS-OCT (Visante OCT, version tions) were evaluated in a masked fashion by a single observer (GB). 2.0; Carl Zeiss Meditec). We enrolled 41 consecutive patients, with 15 Pupil diameter, iris area, and iris thickness in six positions, three from eyes of 15 keratoconus patients and 26 eyes of 26 normal age- and each side of the image (nasal and temporal) were determined (Figu- sex-matched controls (Table 1). For patients and controls, the eye re 1, Table 2) using ImageJ software (version 1.44; National Institutes with the worse BCVA was enrolled. When the BCVA was equivalent in of Health, Bethesda, MD, USA; available at http://rsb.info.nih.gov/ij/ both eyes, the eye with the greatest spherical equivalent refraction index.html). Images were taken directly from the Visante’s output was enrolled. Keratoconus diagnosis was determined by clinical exa- function as RAW files (with lossless data compression). In ImageJ, the mination and corneal modeling with the Pentacam. In accordance file was imported as a 16-bit signed image, with 1024 pixel width and with previous literature(13), keratoconus patients were graded as mild/ 256 pixel height, 0 bytes for offset to first image, 1 for number of ima- moderate (steepest K<52 D) or severe (steepest K>52 D). ges, and 0 bytes for gap between images, preselected as little-endian

306 Arq Bras Oftalmol. 2015;78(5):305-9 Bonfadini G, et al.

byte order. The image was then rotated by 90° to the right position A B and selected in a scale along the x-axis by 8×, maintaining the y-axis scale at 2048 pixel width and 1024 pixel height and bilinear mode in interpolation. With this selection, horizontal scaling was 16 mm/2048 pixels=0.00781 mm/pixel, and vertical scaling was 8 mm/1024 (14) pixel =0.00781 mm/pixel . C The measurements were performed as follows: The left and right SS were defi ned as the point at which a change in curvature of the inner surface of the angle wall became apparent and often presen- ted as inward protrusions of the sclera(15). A perpendicular line was drawn from each left and right SS to the anterior aspect of the band of hyper-refl ectance on the posterior iris surface representing the iris pigment epithelium (Figure 1). From the two ends of these lines, a freehand line was drawn to exactly delineate the anterior iris, central iris border, and posterior edge of the iris stroma as defi ned by the anterior aspect of the band of hyper-refl ectance representing the Figure 1. Anterior segment optical coherence tomography (AS-OCT) images with iris area and iris thickness demarcation. All study images were obtained on the horizontal iris pigment epithelium (Figure 1). The area encircled by this line was meridian. For details regarding the measurement procedure, see the Methods section. A) calculated as the iris area. Left side of the AS-OCT image. B) Right side of the AS-OCT image. C) Full AS-OCT image. The iris cross-sectional area was defi ned as the cross-sectional Iris thicknesses at zones 1 and 6 represent the iris periphery, thicknesses at zones 2 and area of both the nasal and temporal sides. When the left and right 5 represent the iris midpoint, and thicknesses at zones 3 and 4 represent the iris center. ends of the iris are marked in ImageJ, the software provides a cen- troid value for both the left and the right markings. We calculated the horizontal x-axis distance between the two centroids and the vertical y-axis distance between the two centroids in pixels, and then conver- ted them from pixels to mm using scales. This distance was halved Fisher’s exact test. Race-dependent diff erences for each OCT para- to obtain the radius (R), which was then substituted into the formula meter were assessed using a linear mixed-eff ects model. P-values less for iris volume. The mm2/pixel2 value was obtained by multiplying the than 0.05 were considered statistically signifi cant. horizontal scale with the vertical scale: To evaluate the diagnostic performance, receiver operating cha- racteristic (ROC) curves were used to determine the parameter’s overall predictive accuracy [(area under the ROC curve (AROC)) and to identify the optimal iris thickness cutoff points to maximize sensi- tivity and specifi city when discriminating keratoconus patients from ImageJ software provides the area for the left and right iris mar- normal patients. kings. We then converted both areas from pixels2 to mm2 using the scaling factor above; the volume was separately calculated for the two iris markings (180° each) using the following formula: RESULTS The mean age [± standard deviation (SD)] was 38.0 ± 11.4 years , where R is half the distance between (range 18-59 years) in the keratoconus group and 34.1 ± 10.0 years 2 the two centroids in mm and A is the area of the iris in mm . We added (range 20-59 years) in the control group. Of these, 77.7% and 46.2% the two volumes for the left and right iris markings to obtain the fi nal were male in the keratoconus and control groups, respectively. The volume (Zeiss Meditec, personal communication). race distributions in the keratoconus and control groups, respectively, For each portion of the iris in the image (left and right), the midpoint were: white (46.7% vs. 61.5%), black (46.7% vs. 3.9%), Hispanic (6.7% vs. of the line connecting the SS to the iris pigment epithelium was con- 7.7%), and Asian (0.0% vs. 19.2%). The participant characteristics of nected with a line to the central-most location of the pupil margin both groups are shown in table 1. The mean (± SD) spherical equi- (Figure 1, yellow line). This line was divided into four equal portions valent refraction was -2.0 ± 2.4 D in the keratoconus group and -4.8 by drawing three equidistant vertical lines across the iris from the ± 3.0 D in the control group (p=0.004). There was no apparent rela- anterior iris surface to the anterior aspect of the hyper-refl ective band tionship between the iris parameters and severity of disease in the on the posterior iris; the iris thicknesses were obtained by measuring keratoconus patients (mild/moderate severity, eight; severe, seven). between these lines, with zones 1-3 on the left and zones 4-6 on the The mean iris thicknesses for the paired locations (periphery right (Figure 1). zones 1 and 6, midpoint zones 2 and 5, and center zones 3 and 4) Iris thicknesses at zones 1 (left side) and 6 (right side) represent in light and dark are shown in table 2. Compared with controls, the the periphery of the iris; thicknesses at zones 2 (left side) and 5 (right mean iris thicknesses (mm) in light in keratoconus patients (Table 2) side) represent the midpoint of the iris; and thicknesses at zones 3 were 11% thinner for the peripheral iris (zones 1 and 6, p=0.09), 16% (left side) and 4 (right side) represent the center of the iris (Figure 1). thinner for the mid-iris (zones 2 and 5, p=0.02), and 18% thinner for The pupillary diameter was measured as the distance between the the central iris (zones 3 and 4, p=0.004). After adjusting for race, there central-most portions of the iris on the left and right. were signifi cant diff erences for most of the iris parameters and for all three combined zones (1 and 6, 2 and 5, and 3 and 4) in light between StAtiSticAl AnAlySiS keratoconus patients and controls (Table 2). Statistical analysis was performed using Statistical Package for Compared with controls, the mean iris thicknesses (mm) in dark the Social Sciences (SPSS) software version 17.0 (SPSS Inc., Chicago, in keratoconus patients (Table 2) were 19% thinner for the peripheral IL, USA). The demographics, clinical data, and iris parameters (age, sex, iris (zones 1 and 6, p=0.001), 12% thinner for the mid-iris (zones 2 refraction, pupil diameter in dark and light, pupil diameter diff erence and 5, p=0.10), and 15% thinner for the central iris (zones 3 and 4, in dark and light, iris area in dark and light, and iris thickness in dark p=0.004). After adjusting for race, there were signifi cant diff erences and light) were compared between the keratoconus and control for two of the combined zones (1 and 6 and 3 and 4) in dark between groups using two-tailed t-tests. The diff erence in racial distribution keratoconus patients and controls (Table 2). Race adjustment did not between the keratoconus and control groups was assessed using result in a statistically signifi cant diff erence for zones 2 and 5 in dark

Arq Bras Oftalmol. 2015;78(5):305-9 307 Quantitative analysis of iris parameters in keratoconus patients using optical coherence tomography

(p=0.25) between keratoconus patients and controls. be the use of AS-OCT to determine iris parameters predictive of Compared to controls, the total iris area in keratoconus patients keratoconus.­­ (Table 2) was 7% smaller in light (p=0.14) and 10% smaller in dark The human eye is formed through coordinated interactions between (p=0.03). The pupil diameter in light and dark and difference in pupil the neuroepithelium, surface ectoderm, and extraocular mesenchy­ diameter (dark minus light) showed no significant differences in ke- me(20). The neuroectoderm gives rise to the iris, retina, and optic nerve; ratoconus patients compared with controls (Table 2). the surface ectoderm forms the lens and corneal epithelium; and the To discriminate keratoconus cases from controls, the mean iris extraocular mesenchyme, comprising the mesodermal and neural thickness from light and dark conditions in all zones gave an AROC of crest cells, gives rise to the iris, corneal stroma, corneal endothelium, 0.8154, with a maximum sensitivity of 80% using a cutoff of 0.3725 mm extraocular muscles, and fibrous and vascular coats of the eye(21,22). and a maximum specificity of 84.6% using a cutoff of 0.3375 mm The iris arises from the anterior margin of the optic cup neuroepithe- (Table 3). Using the mean iris thickness from light and dark conditions lium and the periocular mesenchyme. The most posterior layer of the in zones 3 and 4 only, the AROC increased to 0.8256, with a maximum iris is the pigmented epithelium; anterior to this are the iris muscles, sensitivity of 86.7% using a cutoff of 0.4700 mm and a maximum spe- and further anteriorly lies the iris stroma. cificity of 84.6% using a cutoff of 0.4125 mm (Table 3 and Figure 2). The iris stroma originates from both the neural crest and meso­ derm(23,24). Similarly, the mesenchymal stromal cells of the neural DISCUSSION crest and mesoderm migrate into the space between the corneal epithelium and endothelium, forming the keratoblasts. The kerato- The utility of AS-OCT in the diagnosis and management of va- (5,6,16-18) blasts proliferate and synthesize an embryonic corneal stroma. The rious anterior segment conditions has been studied in the past . keratoblasts then differentiate into keratocytes, which synthesize a In particular, Prata et al. used AS-OCT to show iris thinning in patients mature stromal extracellular matrix(25). taking systemic alpha-1 adrenergic receptor antagonists associated Although speculative, we suggest that the embryologic simila- with intraoperative floppy iris syndrome(19). In this report, we descri­ rities of time, space, and origin of the iris and corneal stromal cells could explain our observation of reduced iris thickness in keratoco- nus patients. The mechanism could involve defective production of Table 2. Participant iris measurements stromal components, which may be less clinically relevant for the iris Keratoconus Controls than for the cornea, which is under constant biomechanical stress n=15 n=26 P-value due to IOP and contributes directly to visual acuity via refraction and Parameter Mean (SD) Mean (SD) Crude Race adjusted clarity. However, possible abnormalities of the iris structure and bio- mechanics may manifest in our perception of intraoperative floppy Iris thickness (mm) iris syndrome during PKP, as well as the association between Light combined* Urrets-Zavalia syndrome and keratoconus. Zones 1 and 6 0.248 (0.055) 0.279 (0.055) 0.090 <0.030 The findings and implications of this study need to be interpreted Zones 2 and 5 0.296 (0.067) 0.353 (0.073) 0.020 <0.030 with caution because of some limitations, including the small size of the cohort (41 eyes) and the discrepancy between the ethnicities of Zones 3 and 4 0.378 (0.080) 0.463 (0.070) 0.004 <0.001 the keratoconus and control patients, as we enrolled 41 consecutive Dark combined* patients (15 keratoconus patients and 26 controls) on clinic-based Zones 1 and 6 0.300 (0.059) 0.369 (0.063) 0.001 <0.008 evaluation who presented to the cornea service at the Wilmer Eye Zones 2 and 5 0.334 (0.072) 0.379 (0.088) 0.100 <0.250 Institute. Zones 3 and 4 0.381 (0.078) 0.446 (0.060) 0.004 <0.020 In a recent iris thickness study, although Caucasian-Americans

2 had the lowest measurements for all iris thickness parameters, Iris area (mm ) African-Americans had the highest measurements for nearly all iris Light 32.70 (4.20) 35.00 (5.00) 0.140 <0.160 thickness parameters(26). In our study, the results showed that iris Dark 30.90 (4.60) 34.40 (4.60) 0.030 <0.020 thickness was lower in keratoconus eyes than in control eyes, and the Pupil diameter (mm) distribution of our African-American cohort was 46.7% (keratoconus group) and 3.9% (control group). Light 2.45 (0.66) 2.46 (0.66) 0.950 ND Furthermore, the use of six positions under two different light Dark 4.42 (1.04) 4.71 (1.03) 0.400 ND conditions may not reflect the full range of iris abnormalities in Difference (dark-light) 2.00 (0.85) 2.20 (0.85) 0.440 ND keratoconus. Additional possibilities include the use of video recor- SD= standard deviation. dings to capture the dynamic parameters of the iris under changing *= combined indicates the mean, not additive, values for the specified zones. illumination as described by Zheng et al.(27). Furthermore, the ND= not done. cross-sectional design of our study does not address the issue of

Table 3. Diagnostic accuracy of iris parameters for keratoconus diagnosis Parameter Area under ROC Cutoff (mm) Sensitivity (95% CI) Specificity (95% CI) Mean for zones 3 and 4 for both light and dark 0.8256 0.4125 80.0 (51.9-95.7) 84.6 (65.1-95.6) 0.4700 86.7 (59.5-98.3) 61.5 (20.2-59.4) Mean for all zones for both light and dark 0.8154 0.3375 73.3 (44.9-92.2) 84.6 (65.1-95.6) 0.3725 80.0 (51.9-95.7) 61.5 (40.6-79.8) Mean for zones 1 and 6 in dark 0.7910 0.3300 80.0 (51.9-95.7) 69.2 (48.2-85.7) Mean for zones 3 and 4 in light 0.7872 0.4500 80.0 (51.9-95.7) 57.7 (23.3-63.1) ROC= receiver operating characteristic curve; CI= confidence interval.

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